Effect of Proteolytic Enzymes on the Binding of Cobalamin to R Protein and Intrinsic Factor

Allen, Robert H.; Seetharam, Bellur; Podell, Elaine R.; Alpers, David H.

doi:10.1172/jci108924

Cited by 167 publications

(42 citation statements)

References 18 publications

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“…Cbl-analogues were not revealed in cow milk and the amount of TC-Cbl (3 nmol per liter) was quite sufficient to satisfy the demands of daily intake for man (1 nmol) [35]. The instability of TC-Cbl at pH 2 guaranties the liberation of Cbl in acidic medium of human stomach and its binding to human IF and HC [37,38]. It means that cow milk is an appropriate source of B~2 for man.…”

Section: Discussionmentioning

confidence: 99%

Transcobalamin from cow milk: isolation and physico-chemical properties

Fedosov

Petersen

Nexø

1996

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, ieacted with Cbl even in the presence of a B12-analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The Stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displace human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.

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Section: Discussionmentioning

confidence: 99%

Transcobalamin from cow milk: isolation and physico-chemical properties

Fedosov

Petersen

Nexø

1996

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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“…In humans, the genes for IF and HC are located in chromosome 22 (7,8), whereas the gene for TC is located in chromosome 11 (9). Human IF contains 399-aa residues plus Ϸ15% carbohydrate, giving it a molecular mass of Ϸ60 kDa, as observed by gel filtration (10)(11)(12)(13). All three proteins promote Cbl entry through endocytosis involving distinct cell surface receptors (5,(14)(15)(16)(17)(18)(19)(20).…”

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confidence: 99%

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

Mathews¹,

Gordon

Chen³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The structure of intrinsic factor (IF) in complex with cobalamin (Cbl) was determined at 2.6-Å resolution. The overall fold of the molecule is that of an ␣6/␣6 barrel. It is a two-domain protein, and the Cbl is bound at the interface of the domains in a base-on conformation. Surprisingly, two full-length molecules, each comprising an ␣-and a ␤-domain and one Cbl, and two truncated molecules with only an ␣-domain are present in the same asymmetric unit. The environment around Cbl is dominated by uncharged residues, and the sixth coordinate position of Co 2؉ is empty. A detailed comparison between the IF-B12 complex and another Cbl transport protein complex, trans-Cbl-B12, has been made. The pH effect on the binding of Cbl analogues in transport proteins is analyzed. A possible basis for the lack of interchangeability of human and rat IF receptors is presented.5] is a water-soluble vitamin and is essential for the growth and development of all mammals, including humans. Cbl is a member of the corrinoid series of cobalt-containing compounds and is distinguished from some other members of this group by possessing a nucleotide side chain terminating in dimethylbenzimidazole (1, 2). It serves as a key enzymatic cofactor for a number of methyltransferase and mutase reactions occurring in nature (3). In mammals, it contributes the prosthetic group for two important enzymes, methionine synthase (a methyltransferase) and methylmalonylCoA mutase (1, 2, 4).In mammals, three proteins are involved in the uptake, transport, and storage of Cbl. These are gastric intrinsic factor (IF), trans-Cbl II (TC), and haptocorrin (HC). These are immunologically distinct proteins with a protein core of Ϸ46 kDa. IF and HC are heavily glycosylated, but TC is not (5, 6). In humans, the genes for IF and HC are located in chromosome 22 (7,8), whereas the gene for TC is located in chromosome 11 (9). Human IF contains 399-aa residues plus Ϸ15% carbohydrate, giving it a molecular mass of Ϸ60 kDa, as observed by gel filtration (10-13). All three proteins promote Cbl entry through endocytosis involving distinct cell surface receptors (5,(14)(15)(16)(17)(18)(19)(20).Dietary Cbl is bound first to HC in the gastric lumen but is transferred to IF in the duodenum after degradation of HC by pancreatic proteases. IF is responsible for transit of Cbl through the small intestine and delivery of it to the endothelial cells that line the ileum. The IF-Cbl complex is then recognized by the IF-Cbl receptor, cubilin (CUB), located on the luminal side of the intestinal mucosal cells, and mediates its internalization. Lysosomal degradation of the internalized IF-Cbl complex releases Cbl, which is then able to bind to TC, probably within the enterocyte. The TC-Cbl complex is then translocated across the basolateral membrane and released into the circulation, to be taken up by TC-Cbl receptor-mediated process by all cells in the body. Lysosomal dissociation of the complex then occurs along with transfer of Cbl to the coenzyme methyl-and Ado-Cbl in the cytoplasm and i...

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“…In addition, other concurrent (14) and more recent in vivo studies (17) demonstrated that IF free of Cbl remains biologically, immunologically, and physicochemically unaltered, whereas the R protein(s) are being degraded during normal intraluminal transport. These latter observations supported the thesis (16) that in EPI the R protein(s) are not subject to degradation and therefore the entire fraction of Cbl bound to these nonfunctional proteins will not be absorbed (14,16,17). Recent studies in Rothenberg's laboratory (18) indicated that bile effectively sequesters Cbl from IF to nonfunctional binders and that this process can be reversed after treatment with proteases (18).…”

Section: Introductionmentioning

confidence: 58%

“…It appears likely: (a) that the impairment of Cbl absorption in EPI is a result of a biochemical defect that supervenes somewhere between the gastric and the ileal wall phase of the Cbl transport, i.e., during the intraluminal phase of Cbl transport and; (b) that this defect does not alter the function of IF as such but rather the mode of transport of Cbl itself. These possibilities have recently been fully appreciated by Allen et al (16), who speculated that the presence of undegraded R-type Cbl-binding protein(s) (R protein,2 cobalophilin, non-IF, etc.) in the intestinal lumen can inhibit the ileal absorption of the vitamin, and showed by in vitro studies that purified pancreatic proteases can degrade R proteins but not IF (16).…”

Section: Introductionmentioning

confidence: 99%

“…These possibilities have recently been fully appreciated by Allen et al (16), who speculated that the presence of undegraded R-type Cbl-binding protein(s) (R protein,2 cobalophilin, non-IF, etc.) in the intestinal lumen can inhibit the ileal absorption of the vitamin, and showed by in vitro studies that purified pancreatic proteases can degrade R proteins but not IF (16). In addition, other concurrent (14) and more recent in vivo studies (17) demonstrated that IF free of Cbl remains biologically, immunologically, and physicochemically unaltered, whereas the R protein(s) are being degraded during normal intraluminal transport.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cobalamin malabsorption due to nondegradation of R proteins in the human intestine. Inhibited cobalamin absorption in exocrine pancreatic dysfunction.

Marcoullis¹,

Parmentier²,

Nicolas³

et al. 1980

J. Clin. Invest.

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A B S T R A C T In vivo studies demonstrate that the pancreatic enzymes and the ionic environment in the upper gastrointestinal tract are essential determining factors for transport and absorption of cobalamin in man.Jejunal fluid was aspirated from healthy human volunteers after administration of cyano[57Co]cobalamin preparations. Immunochemical analysis of the aspirates demonstrated that all isotopic vitamin was transferred to a protein that is identical to the gastric intrinsic factor in terms of molecular mass (57,500), ionic nature (mean pI, 5.09), and reactivity with antiintrinsic factor sera. However, in the aspirates from patients with exocrine pancreatic dysfunction the vitamin was found to be coupled >60% to a protein identical to R proteins in terms of molecular mass (125,000), ionic nature (mean pI, 3.51), and reactivity with anti-R protein and anti-intrinsic factor sera. The preferential transfer of cobalamin to R proteins in the patients and to intrinsic factor in healthy subjects was associated, respectively, with low and normal levels ofpancreatic enzymes in the intestine and these in turn were paralleled respectively by impaired and normal ileal absorption of cobalamin. These findings confirm the suggestion that the formation of unabsorbable cobalamin complexes may be the reason of impaired vitamin absorption in exocrine pancreatic insufficiency.This paper was presented in part at the 3rd European Symposium on Vitamin B12 and Intrinsic Factor, March 1979, Zurich, Switzerland.Dr. Marcoullis is a visiting professor of medicine under the auspices of Institut National de la Sante et de la Recherch6 Medicale at the University of Nancy. The permanent address for Dr. Marcoullis and Ms. Jimenez is Department of Medicine, Veterans Administration Hospital, Brooklyn, N. Y. Received for publication 2 November 1979 and in revised form 19 May 1980. 430 Observations made with other selected patients demonstrate: (a) that decreased enzyme activity and nondegradation of R proteins may also be due to nonactivation ofpancreatic zymogens in an acidic pH ofthe intestinal juice and; (b) that in the absence of an acidic environment in gastric juice the vitamin transported to the jejunum couples to intrinsic factor when pancreatic function is normal, and to intrinsic factor and R protein in exocrine pancreatic insufficiency. The observations made with these selected patients may explain why not all patients with exocrine pancreatic insufficiency develop impaired cobalamin absorption, and also why the malabsorption is corrected by the administration of bicarbonate in certain patients. INTRODUCTIONA common feature of the untreated exocrine pancreatic insufficiency (EPI)l is the impaired ileal absorption of cobalamin (Cbl; vitamin B,2) (1-8). The mechanism(s) underlying the pathophysiology of this phenomenon have not been worked out (8-12). The two principal prerequisites for normal Cbl absorption i.e., the complexing of dietary Cbl to the gastric intrinsic factor (IF) (gastric phase of Cbl transport), as well as th...

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Effect of Proteolytic Enzymes on the Binding of Cobalamin to R Protein and Intrinsic Factor

Abstract: A B S T R A C T Cobalamin (

Cited by 167 publications

References 18 publications

Transcobalamin from cow milk: isolation and physico-chemical properties

Transcobalamin from cow milk: isolation and physico-chemical properties

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

Cobalamin malabsorption due to nondegradation of R proteins in the human intestine. Inhibited cobalamin absorption in exocrine pancreatic dysfunction.

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